A modified calcium retention capacity assay clarifies the roles of extra- and intracellular calcium pools in mitochondrial permeability transition pore opening

Calcium homeostasis is essential for cell survival and is precisely controlled by several cellular actors such as the sarco/endoplasmic reticulum and mitochondria. Upon stress induction, Ca 2+ released from sarco/endoplasmic reticulum stores and from extracellular Ca 2+ pools accumulates in the cytosol and in the mitochondria. This induces Ca 2+ overload and ultimately the opening of the mitochondrial permeability transition pore (mPTP), promoting cell death. Currently, it is unclear whether intracellular Ca 2+ stores are sufficient to promote the mPTP opening. Ca 2+ retention capacity (CRC) corresponds to the maximal Ca 2+ uptake by the mitochondria before mPTP opening. In this study, using permeabilized cardiomyocytes isolated from adult mice, we modified the standard CRC assay by specifically inducing reticular Ca 2+ release to investigate the respective contributions of reticular Ca 2+ and extracellular Ca 2+ to mPTP opening in normoxic conditions or after anoxia–reoxygenation. Our experiments revealed that Ca 2+ released from the sarco/endoplasmic reticulum is not sufficient to trigger mPTP opening and corresponds to ∼50% of the total Ca 2+ levels required to open the mPTP. We also studied mPTP opening after anoxia–reoxygenation in the presence or absence of extracellular Ca 2+ . In both conditions, Ca 2+ leakage from internal stores could not trigger mPTP opening by itself but significantly decreased the CRC. Our findings highlight how a modified CRC assay enables the investigation of the role of reticular and extracellular Ca 2+ pools in the regulation of the mPTP. We propose that this method may be useful for screening molecules of interest implicated in mPTP regulation.